Bottom Line:
The resulting ICL mutant library was analyzed by biochemical and phenotypic mapping.Based on a 3D homology model and kinetic analysis of drug transport, our data suggest that large distances between ICL residues and their respective chromosomal suppressor mutations rule out a direct interaction between them.However, they impact the transport cycle by restoring the coupling interface via indirect downstream signaling.

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

ABSTRACTThe ABC transporter Cdr1 protein (Cdr1p) of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) that are interconnected by extracellular (ECLs) and intracellular (ICLs) loops. To examine the communication interface between the NBDs and ICLs of Cdr1p, we subjected all four ICLs to alanine scanning mutagenesis, replacing each of the 85 residues with an alanine. The resulting ICL mutant library was analyzed by biochemical and phenotypic mapping. Only 18% of the mutants from this library displayed enhanced drug susceptibility. Most of the drug-susceptible mutants displayed uncoupling between ATP hydrolysis and drug transport. The two drug-susceptible ICL1 mutants (I574A and S593A) that lay within or close to the predicted coupling helix yielded two chromosomal suppressor mutations that fall near the Q-loop of NBD2 (R935) and in the Walker A motif (G190) of NBD1. Based on a 3D homology model and kinetic analysis of drug transport, our data suggest that large distances between ICL residues and their respective chromosomal suppressor mutations rule out a direct interaction between them. However, they impact the transport cycle by restoring the coupling interface via indirect downstream signaling.

f9: View of the Cdr1p TMDs in closed conformation from cytoplasm.All Cdr1p ICLs are shown in yellow. All total drug susceptible mutations determined previously23 in TMDs are shown in blue with TMS1-6 (designated as TM1-6) shown in orange while TMS7-12 (designated as TM7-12) shown in cyan. Two drug-susceptible ICL1 mutations, I574 and S593, which have suppressor mutations in NBDs, are shown in pink. Other drug-susceptible ICL residues in stick model are shown in red and may be involved directly in drug binding interactions of Cdr1p.

Mentions:
Our study also suggests that not all ICL residues have a direct role in interfacial communication. Because ICLs are the extensions of TMSs, and many of the ICL residues located at these cytosolic extensions also fall closer to the drug binding pocket, they could thus impact the binding or release of the drug directly rather than acting as interfacial residues (Fig. 9). In particular, this phenomenon may be possible in drug-susceptible mutants, as depicted in the deduced stick model (red in Fig. 9) in which, the faces are closer to drug binding pocket. Therefore, the loss of transport ability in these variants could be due to a defect in the binding or release of the drug rather than the disruption of inter-domain communication. This possibility needs to be examined in more detail. Finally, identifying uncoupling in a system where the binding of drugs does not cause an enhanced rate of ATPase activity, perhaps due to the presence of endogenous substrates, still remains a challenge. Nevertheless, this study provides an initial insight into the role of ICLs in the activity of a medically important ABC transporter Cdr1p, which could help to improve therapeutic strategies against Candida infections.

f9: View of the Cdr1p TMDs in closed conformation from cytoplasm.All Cdr1p ICLs are shown in yellow. All total drug susceptible mutations determined previously23 in TMDs are shown in blue with TMS1-6 (designated as TM1-6) shown in orange while TMS7-12 (designated as TM7-12) shown in cyan. Two drug-susceptible ICL1 mutations, I574 and S593, which have suppressor mutations in NBDs, are shown in pink. Other drug-susceptible ICL residues in stick model are shown in red and may be involved directly in drug binding interactions of Cdr1p.

Mentions:
Our study also suggests that not all ICL residues have a direct role in interfacial communication. Because ICLs are the extensions of TMSs, and many of the ICL residues located at these cytosolic extensions also fall closer to the drug binding pocket, they could thus impact the binding or release of the drug directly rather than acting as interfacial residues (Fig. 9). In particular, this phenomenon may be possible in drug-susceptible mutants, as depicted in the deduced stick model (red in Fig. 9) in which, the faces are closer to drug binding pocket. Therefore, the loss of transport ability in these variants could be due to a defect in the binding or release of the drug rather than the disruption of inter-domain communication. This possibility needs to be examined in more detail. Finally, identifying uncoupling in a system where the binding of drugs does not cause an enhanced rate of ATPase activity, perhaps due to the presence of endogenous substrates, still remains a challenge. Nevertheless, this study provides an initial insight into the role of ICLs in the activity of a medically important ABC transporter Cdr1p, which could help to improve therapeutic strategies against Candida infections.

Bottom Line:
The resulting ICL mutant library was analyzed by biochemical and phenotypic mapping.Based on a 3D homology model and kinetic analysis of drug transport, our data suggest that large distances between ICL residues and their respective chromosomal suppressor mutations rule out a direct interaction between them.However, they impact the transport cycle by restoring the coupling interface via indirect downstream signaling.

Affiliation:
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

ABSTRACTThe ABC transporter Cdr1 protein (Cdr1p) of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) that are interconnected by extracellular (ECLs) and intracellular (ICLs) loops. To examine the communication interface between the NBDs and ICLs of Cdr1p, we subjected all four ICLs to alanine scanning mutagenesis, replacing each of the 85 residues with an alanine. The resulting ICL mutant library was analyzed by biochemical and phenotypic mapping. Only 18% of the mutants from this library displayed enhanced drug susceptibility. Most of the drug-susceptible mutants displayed uncoupling between ATP hydrolysis and drug transport. The two drug-susceptible ICL1 mutants (I574A and S593A) that lay within or close to the predicted coupling helix yielded two chromosomal suppressor mutations that fall near the Q-loop of NBD2 (R935) and in the Walker A motif (G190) of NBD1. Based on a 3D homology model and kinetic analysis of drug transport, our data suggest that large distances between ICL residues and their respective chromosomal suppressor mutations rule out a direct interaction between them. However, they impact the transport cycle by restoring the coupling interface via indirect downstream signaling.